Microtubule-based motor complexes drive and regulate the intracellular distribution of countless cargos, ranging from organelles and vesicles to mRNAs. While clearly essential for all eukaryotic cells, nowhere is this process more crucial than in neurons, where intracellular transport deficiencies can underly devastating neuro- degenerative conditions. Several theories have been vying to explain how kinesins and dyneins, molecular motors that pull cargos towards opposite poles of microtubules, work with and/or against each other to achieve regulated bi-directional transport. A growing number of studies point to a constant tug-of-war wherein kinesins and dyneins are simultaneously active and working against each other, though this model has never been directly proven in living cells. An ideal strategy to pick apart the contributions of individual motors within the complex would be to photo-inactivate them, then observe the immediate aftermath on moving cargos. However, since such a method for in vivo photo-inactivation doesn't exist in today's molecular toolbox, we set out to create it. As a result, we have developed a small-molecule dimerizer that can be photo-cleaved with targeted laser light. The novel compound, which we have named zapalog, can be used to dimerize any two recombinant proteins/peptides tagged with the zapalog- binding domains, and this dimerization can then be immediately reversed by zapalog photo-cleavage. We propose to verify, characterize and optimize this novel compound, then use it to adhere engineered Kinesin-1 motor domains to their corresponding cargo binding domains in cultured rat neurons and Aspergillas nidulans, effectively re-creating functional kinesin molecules that are susceptible to photo-destruction inside living cells. The ability to instantaneously remove the kinesins from moving cargo affords us a unique position to finally test the tug-of-war model in vivo, by observing the immediate aftermath of kinesin loss on motor complex behavior in living cells. Does a cargo moving anterograde simply stop or does it promptly reverse direction as the dynein motor is no longer opposed? Does a retrogradely moving cargo continue as before or are its speed and processivity suddenly enhanced by the absence of opposing kinesins? Beyond providing an answer to the illusive tug-of-war model, this new technique could become a very valuable tool for a broad range of cell biology questions, as it can be used to integrate in vivo photo-inactivation as well as photo-activation modalities into almost any protein of interest.